Various methods and compositions for targeted cleavage of genomic DNA have been described. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination at a predetermined chromosomal locus. See, e.g., U.S. Pat. Nos. 9,255,250; 9,200,266; 9,045,763; 9,005,973; 9,150,847; 8,956,828; 8,945,868; 8,703,489; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060063231; 20080159996; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983; 20130196373; 20140120622; 20150056705; 20150335708; 20160030477 and 20160024474, the disclosures of which are incorporated by reference in their entireties for all purposes.
These methods often involve the use of engineered cleavage systems to induce a double strand break (DSB) or a nick in a target DNA sequence such that repair of the break by an error born process such as non-homologous end joining (NHEJ) or repair using a repair template (homology directed repair or HDR) can result in the knock out of a gene or the insertion of a sequence of interest (targeted integration). Cleavage can occur through the use of specific nucleases such as engineered zinc finger nucleases (ZFN), transcription-activator like effector nucleases (TALENs), using the CRISPR/Cas system (including Cas and/or Cfp1) with an engineered crRNA/tracr RNA (‘single guide RNA’) to guide specific cleavage and/or using nucleases based on the Argonaute system (e.g., from T. thermophilus, known as ‘TtAgo’, (Swarts et at (2014) Nature 507(7491): 258-261).
Targeted cleavage using one of the above mentioned nuclease systems can be exploited to insert a nucleic acid into a specific target location using either HDR or NHEJ-mediated processes. However, delivering both the nuclease system and the donor to the cell can be problematic. For example, delivery of a donor or a nuclease via transduction of a plasmid into the cell can be toxic to the recipient cell, especially to a cell which is a primary cell and so not as robust as a cell from a cell line.
One method often utilized for delivery of nucleic acids to cells involves the use of viral nucleic acid delivery vectors. In particular, the adeno associated virus (AAV) is widely used to deliver nucleic acid because of its efficiency and relative non-toxicity. The AAV genome can be nearly depleted of viral nucleic acid and replaced with nucleic acids encoding donor transgenes or engineered nucleases to facilitate integration of the transgene into a recipient cell's DNA.
AAV transduction of mammalian cells depends on both primary and secondary co-receptors on the target cells. While the primary receptor is important for initial adhesion of the virus to the target cell (and its tropism), the secondary receptor mediates endocytosis of the AAV virus into the cell. For example, for serotype AAV6 the primary receptor has been identified as alpha 2,3 N-linked sialic acid (Wu et al, (2006) J. Virol. 80(18):9093), and the secondary receptor as EGFR. Furthermore, the use of additional secondary co-receptors has also been proposed (Weller et al, (2010) Nat Med 16(6): 662).
Delivery (transplantation) of cells and/or tissues in vivo can often be hampered by antibody-mediated responses. For example, some kidney transplant patients are prone to acute rejection mediated by the development of host antibodies against the transplant tissue. Accordingly, physicians routinely use steroid therapy to suppress the antibody response following transplantation (see for example Ku et at (1973) Br. Med J 4:702) and also can use rituximab (anti-CD20 antibody) for B cell suppression (for example Becker et at (2004) Am J Transpl 4:996). Antibody-mediated responses are also challenges facing the use of AAV delivery due to prevalence of background anti-AAV antibodies in the human population and the de novo development of these antibodies following dosing with a AAV mediated delivery system (see Kotterman et at (2015) Gene Ther 22(2):116-126).
In the body, there are complex mechanisms that can regulate either the activation or the suppression of the cellular members of the immune system. For example, dendritic cells (DCs) have been established as central players in the balance between immune activation versus immune tolerance. They are the most potent antigen presenting cells in the immune system and specifically capture and present antigens to naïve T cells. Immature DCs interact with potential antigens through specific receptors such as Toll-like receptors where the antigen is brought into the cell by micropinocytosis. The antigen is then broken up into smaller peptides that are presented to T cells by the major histocompatibility complexes. In addition, mature DCs secrete inflammatory mediators such as IL-1β, IL-12, IL-6 and TNF which further serve to activate the T cells. On the other side, DCs also play a role in tolerizing the body to some antigens in order to maintain central and peripheral tolerance. Tolerogenic DCs (tolDC) have low amounts of co-stimulatory signals on the cell surfaces and have a reduced expression of the inflammatory mediators described above. However, these tolDCs express large amounts of anti-inflammatory cytokines like IL-10 and when these cells interact with naïve T cells, the T cells are driven to become anergic/regulatory T cells (CD8+ Tregs). In fact, it has been shown that this process is enhanced upon repeated stimulation of T cells with these immature/tolerogenic DCs. Several factors have also been identified that work in concert with tolDCs to induce different types of Tregs. For example, naïve T cells co-exposed with tolDCs and HGF, VIP peptide, TSLP or Vitamin D3 leads to the induction of CD4+CD25+Foxp3+ Tregs, co-exposure with TGF-β or IL-10 leads to Tr1 T regs and co-exposure with corticosteroids, rapamycin, retinoic acid can lead to CD4+/CD8+ Tregs (Raker et at (2015) Front Immunol 6: art 569 and Osorio et at (2015) Front Immunol 6: art 535).
CD34+ stem or progenitor cells are a heterogeneous set of cells characterized by their ability to self-renew and/or differentiate into the cells of the lymphoid lineage (e.g. T cells, B cells, NK cells) and myeloid lineage (e.g. monocytes, erythrocytes, eosinophiles, basophiles, and neutrophils). Their heterogeneous nature arises from the fact that within the CD34+ stem cell population, there are multiple subgroups which often reflect the multipotency (whether lineage committed) of a specific group. For example, CD34+ cells that are CD38− are more primitive, immature CD34+ progenitor cell, (also referred to as long term hematopoietic progenitors), while those that are CD34+CD38+ (short term hematopoietic progenitors) are lineage committed (see Stella et at (1995) Hematologica 80:367-387). When this population then progresses further down the differentiation pathway, the CD34 marker is lost. CD34+ stem cells have enormous potential in clinical cell therapy. However, in part due to their heterogeneous nature, performing genetic manipulations such as gene knock out, transgene insertion and the like upon the cells can be difficult. Specifically, these cells are poorly transduced by conventional delivery vectors, the most primitive stem cells are sensitive to modification, there is limited HDR following induced DNA DSBs, and there is insufficient HSC maintenance in prolonged standard culture conditions. Additionally, other cells of interest (for non-limiting example only, cardiomyocytes, medium spiny neurons, primary hepatocytes, embryonic stem cells, induced pluripotent stem cells and muscle cells) can be less successfully transduced for genome editing than others.
Thus, there remains a need for additional compositions and methods for genome engineering to deliver nucleic acids efficiently to CD34+ cells and other cells of interest using viral vectors.